Efficacy of urolithin B-Loaded mesoporous silica nanoparticles modified with graphene quantum Dots against MCF-7 breast Cancer cells

Breast cancer remains one of the most prevalent malignancies among women worldwide, posing a significant challenge to global healthcare systems. According to the World Health Organization (WHO), approximately 2.3 million women were diagnosed with breast cancer in 2022, making it the most commonly diagnosed cancer globally¹. Among the various subtypes, MCF-7 breast cancer cells, which are estrogen receptor-positive (ER+), have been extensively studied due to their clinical relevance and responsiveness to targeted therapies². This cell line is crucial for studying breast cancer biology and testing new drugs, as ER + tumors make up about 70% of all cases³. Despite significant advancements in treatment strategies, including surgery, chemotherapy, radiotherapy, and hormone therapy, the shortcomings of these methods make the treatment of advanced and metastatic breast cancer still challenging. Challenges such as drug resistance and tumor heterogeneity limit the effectiveness of traditional therapies^4,5. These limitations highlight the urgent need for innovative therapeutic approaches to enhance drug efficacy, reduce side effects, and overcome resistance mechanisms. Ongoing research and clinical trials are essential for developing new treatment strategies to enhance patients’ quality of life with this devastating disease.

Nanotechnology has emerged as a powerful tool in cancer therapy, offering promising solutions to overcome the limitations of conventional treatments. One of the key advancements in this field is the development of nanocarriers, which can significantly enhance the solubility, stability, and targeted delivery of anti-cancer drugs. This, in turn, improves their therapeutic index while minimizing adverse effects^4,6. In breast cancer treatment, nanotechnology-based drug delivery systems have demonstrated improved cellular uptake, controlled drug release, and bypassing multidrug resistance mechanisms, critical for effective therapy. Various nanomaterials, including liposomes, polymeric nanoparticles, and inorganic nanocarriers, have been explored to deliver chemotherapeutic agents and natural compounds with enhanced efficacy.

Among these, mesoporous silica nanoparticles (MSNs) have garnered considerable attention as versatile nanocarriers due to their high surface area, tunable pore size, biocompatibility, and ability to encapsulate a wide range of therapeutic agents^7,8. Modifying MSNs with functional nanomaterials, such as polymers and targeting ligands, boosts their therapeutic potential by improving cellular uptake and specificity⁹. Graphene quantum dots (GQDs), a novel class of carbon-based nanomaterials, possess excellent physicochemical properties, including high photostability, biocompatibility, and fluorescence, which can be exploited to enhance drug delivery systems¹⁰. Their zero-dimensional structure and high surface-to-volume ratio enhance interaction with biological systems, making them suitable for biomedical applications¹¹. Incorporation of GQDs into MSNs not only facilitates targeted delivery but also contributes to synergistic anti-cancer effects through reactive oxygen species (ROS) generation, promoting apoptosis in cancer cells^12,13. This multifunctionality makes GQD-modified MSNs a promising platform for breast cancer therapy.

Urolithin B (Uro-B), a metabolite derived from ellagitannins found in pomegranates and certain nuts, has attracted significant interest due to its potent anti-cancer properties¹⁴. Uro-B induces apoptosis, inhibits proliferation, and modulates critical signaling pathways such as PI3K/Akt and MAPK, which regulate cell survival and growth¹⁵. It can alter the tumor microenvironment, influencing inflammatory cytokines and promoting autophagy, which may help prevent cancer progression^16,17. These attributes position Uro-B as a promising therapeutic candidate for breast cancer. However, its poor water solubility and low bioavailability limit its clinical application¹⁸. To address these challenges, nanocarrier-based delivery systems are being developed to enhance Uro-B’s solubility, stability, and targeted delivery. In this study, we developed a nanosystem comprising Uro-B-loaded MSNs modified with GQDs (GQD@MSN-Uro-B) to improve the therapeutic efficacy against MCF-7 breast cancer cells. We evaluated its cytotoxicity, cellular uptake, and underlying anti-cancer mechanisms, aiming to demonstrate the potential of this nanoplatform as an effective and selective treatment strategy for breast cancer.

Chemicals

Graphite powder (≥ 99.9%), potassium nitrate (KNO₃, ≥ 99%), sulfuric acid (H₂SO₄, 98%), potassium permanganate (KMnO₄, ≥ 99%), hydrogen peroxide (H₂O₂, 30%), and dimethylformamide (DMF, ≥ 99.8%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cetyltrimethylammonium bromide (CTAB, ≥ 99%), tetraethyl orthosilicate (TEOS, 98%), and ammonium hydroxide (NH₄OH, 28–30%) were obtained from Merck (Darmstadt, Germany). Golexir Pars Company, School of Pharmacy, Ferdowsi Campus, Mashhad, Iran, supplied Urolithin B (Uro-B, purity ≥ 98%). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, ≥ 98%) and N-hydroxysuccinimide (NHS, ≥ 98%) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ethanol (analytical grade), phosphate-buffered saline (PBS), and methanol (analytical grade) were sourced from Scharlau (Barcelona, Spain).

Preparation of GQDs

Graphene oxide (GO) was synthesized from 2 g of graphite powder and 1 g of KNO₃ mixed with 100 mL of 98% H₂SO₄ at 0 °C. KMnO₄ (15 g) was gradually added, keeping the temperature below 20 °C, followed by heating at 40 °C for 24 h. After adding 50 mL of deionized water, the temperature was raised to 98 °C. Upon cooling, the mixture was diluted with 250 mL of deionized water, and 10 mL of H₂O₂ was added until bubbling ceased. The solution was centrifuged, washed until neutral, and dialyzed. The resulting GO was ultrasonicated using a Branson 450 Sonifier probe sonicator at an amplitude of 30% for 24 h, with temperature monitored to ensure it remained below 40 °C by pausing intermittently for cooling. After ultrasonication, the GO was centrifuged at 3000 rpm and freeze-dried for 2 days. To synthesize GQDs, 150 mg of GO was dissolved in 17 mL of dimethylformamide (DMF) and sonicated for 30 min. The mixture was heated in a Teflon-lined autoclave at 200 °C for 5 h, centrifuged, and the solvent evaporated. The solid was dialyzed in deionized water to obtain a GQD suspension¹⁹.

Synthesis of MSN

MSNs were synthesized using a sol-gel process that involves the silica precursor tetraethyl orthosilicate (TEOS) and the surfactant cetyltrimethylammonium bromide (CTAB). The procedure began by dissolving 75 mg of CTAB in 30 mL of deionized water at a temperature of 45 °C. Next, 125 µL of TEOS was added under basic conditions, which were created by adding 275 µL of ammonium hydroxide (NH₄OH) to catalyze the polymerization of silica. The mixture was stirred for 2 h at a speed of 300 rpm to allow the formation of silica structures around the surfactant micelles. The particles were collected by centrifugation once the desired morphology and pore size were achieved. They were then washed with 1% acidic methanol at 60 °C for 4 h to remove the CTAB and create mesopores. After washing, the particles were dried, and finally, calcination was performed at high temperatures. This synthesis process yields highly uniform, porous silica nanoparticles suitable for various applications, including drug delivery, catalysis, and sensing.

Synthesis of GQD-NHS-EDC-MSN

To activate carboxyl groups on GQDs using EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)/NHS (N-hydroxysuccinimide), a solution of GQDs was first prepared in phosphate-buffered saline (PBS) at a concentration of 2 mg/mL. EDC and NHS were dissolved in the same buffer in a separate vial to achieve final concentrations of 0.1 M and 0.05 M, respectively. The EDC/NHS solution was then added to the GQD suspension, and the mixture was gently mixed to ensure uniform distribution. The mixture was incubated at room temperature for 60 min to allow activation of the carboxyl groups. After activation, MSNs were added to the GQD solution, and the mixture was stirred for 2 h to facilitate covalent bonding between the activated GQDs and MSNs. Finally, the resulting conjugates were purified by centrifugation or dialysis to remove unreacted reagents and obtain stable GQD-MSN conjugates²⁰.

Loading of Uro-B onto GQDs-MSN

Two methods were employed for loading Uro-B onto the nanomaterials. In Method 1, a solution of Uro-B was prepared in ethanol and mixed with GQDs-MSN to create a uniform suspension. The mixture was ultrasonicated for 30 min and stirred at 200 rpm for 24 h at room temperature to facilitate Uro-B loading onto the GQD-MSNs. After this, the mixture was centrifuged to separate unbound Uro-B, and the pellet was washed with ethanol to remove any excess drug. In Method 2, MSNs were first loaded with Uro-B, allowing the drug to fill the pores and adhere to the surface. After achieving the desired loading, the drug-loaded MSNs were coated with GQDs, ensuring that Uro-B was effectively encapsulated within the pores and on the surface of the MSNs. For clarity, the formulations were designated as GQD@MSN-Uro-B for Method 1 and MSN-Uro-B@GQD for Method 2. Additionally, the encapsulation efficiency of GQDs-MSN was calculated using the formula: (weight of Uro-B/ weight of GQDs-MSN) × 100%. Finally, the loaded GQDs-MSN were resuspended in an appropriate buffer for further characterization.

Drug release assay

The drug release profile of Resveratrol from MWCNT-Chi-FA-Resveratrol NPs was evaluated by immersing a specific amount of the formulation in 10 ml of PBS (pH 7.4) at 37 °C. Samples were taken at predetermined intervals (10, 20, 30, 40, 50, 60, and 70 h) and centrifuged at 10,000 rpm for 10 min to separate the released Resveratrol from the MWCNT-Chi-FA-Resveratrol NPs. The supernatant was then analyzed using a UV spectrophotometer at 321 nm to quantify the amount of Resveratrol released. The cumulative release percentage was calculated based on the initial drug content, allowing for assessing the release kinetics over time.

NP characterization

Given the importance of particle size in drug delivery systems (DDSs), dynamic light scattering (DLS) was employed to analyze particles’ hydrodynamic size and distribution in an aqueous solution. This technique facilitates particle size distribution and stability assessment while in suspension. Additionally, field emission scanning electron microscopy (FESEM) was used to evaluate the morphology of the particles, and Fourier-transform infrared (FT-IR)spectroscopy was performed to investigate the functional groups present²¹.

Cytotoxicity assay

The MTT assay was conducted to evaluate the cytotoxic effects of drug-loaded NPs on MCF7 cancer cell lines and human dermal fibroblast (HDF) cells. Initially, both cell types were seeded at a density of 1 × 10⁴ cells in 96-well plates and allowed to adhere for 24 h. The cells were treated with various concentrations of NPs (7.8, 15.6, 31.2, 62.5, 125, 250, and 500 µg/mL) for 48 h. After the treatment, 20 µL of MTT solution (5 mg/mL) was added to each well and incubated for 4 hours at 37 °C. Following incubation, the culture medium was removed, and the formazan crystals were dissolved in 200 µL of dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm using a microplate reader, and cell viability was calculated as a percentage of the control group, allowing for the assessment of the cytotoxic effects of the NPs on both cell lines. Cell viability was calculated as a percentage of the control, and IC₅₀ values, the concentration of drug required to inhibit cell viability by 50%, were determined for each cell line.

Annexin V-FITC/PI assay

To evaluate the apoptotic effects of GQD@MSN-Uro-B NPs on MCF7 cells, the Annexin V-FITC/propidium iodide (PI) dual staining assay was performed. MCF7 cells were cultured at a density of 1 × 104 cells per well for 24 h and then treated with GQD@MSN-Uro-B NPs at concentrations of 8, 10, and 12 µg/mL for 24 h. The 10 µg/mL concentration was determined as the IC₅₀, while the 8 and 12 µg/mL concentrations were chosen to induce approximately 25% and 75% cell death, respectively. After treatment, the cells were harvested, washed with PBS, and resuspended in a binding buffer at a concentration of 1 × 10⁶ cells/mL. Subsequently, Annexin V-FITC (5 µL) and PI (5 µL) were added, and the cells were incubated for 15 min in the dark. The stained cells were analyzed using flow cytometry to quantify the percentage of early apoptotic (Annexin V+, PI-), late apoptotic (Annexin V+, PI+), and necrotic (Annexin V-, PI+) cells.

Acridine orange/propidium iodide (AO/PI) staining

To evaluate apoptosis in MCF7 cells treated with GQD@MSN-Uro-B NPs, AO/PI staining was employed. MCF7 cells were cultured at a density of 1 × 10⁴ cells per well in 6-well plates and exposed to the 8, 10, and 12 µg/mL concentrations of GQD@MSN-Uro-B NPs for 48 h. After treatment, the cells were trypsinized, washed with PBS, and resuspended in PBS with acridine orange (1 µg/mL) and propidium iodide (5 µg/mL) for 10 min at room temperature. They were then rewashed and examined under a fluorescence microscope (Carl Zeiss, Jena, Germany).

Real-time polymerase chain reaction (real-time PCR).

Real-time PCR was performed to analyze caspase 9, TNF, and P21 mRNA expression levels in MCF7 cells. MCF7 cells were cultured and treated with GQD@MSN-Uro-B NPs at 8, 10, and 12 µg/mL for 48 h. Following treatment, total RNA was extracted using an RNA extraction kit according to the manufacturer’s instructions (QIAGEN). The quality and quantity of the extracted RNA were assessed using a ThermoFisher Nanodrop. Subsequently, complementary DNA (cDNA) was synthesized from 1 µg of RNA using a reverse transcription kit (Promega). Real-time PCR was conducted using specific primers (Table 1) for caspase 9, TNF, and P21, along with a housekeeping gene (GAPDH) as a control. The PCR reactions were carried out in triplicate, and the relative expression levels of the target genes were quantified using the ΔΔCT method. The results provided insights into the regulatory roles of these genes in the apoptotic process induced by GQD@MSN-Uro-B NPs in MCF7 cells.

Antioxidant activity assay

The ABTS and DPPH assays were conducted to evaluate the antioxidant capacity of GQD@MSN-Uro-B NPs. For the ABTS assay, a stock solution of ABTS was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate and allowing it to stand in the dark for 12 h. The resulting ABTS radical cation was diluted with PBS to an absorbance of 0.70 at 734 nm. Various concentrations of GQD@MSN-Uro-B NPs were mixed with the diluted ABTS solution and incubated for 6 min before measuring the absorbance at 734 nm. For the DPPH assay, a 0.1 mM DPPH solution was prepared in methanol. Various concentrations of the NPs were added to the DPPH solution and incubated in the dark for 30 min. The absorbance was measured at 517 nm, and the percentage of DPPH radical scavenging was calculated. The radical scavenging activity was calculated using the formula: % inhibition = (control absorbance – sample absorbance) / control absorbance × 100.

Statistical analysis

Statistical analyses were performed using SPSS software (version 22). The normality of the data was assessed with the Shapiro-Wilk test. A one-way analysis of variance (ANOVA) was utilized to compare differences among multiple groups, and Tukey’s post hoc test was applied for detailed pairwise comparisons to identify specific group differences. A significance level of p < 0.05 was set. Results are reported as means ± standard deviations (SD).

This study aimed to evaluate the anti-cancer effects of GQD@MSN-Uro-B nanoparticles on the MCF7 cancer cell line while assessing their antioxidant properties.

Encapsulation efficacy and drug release assay

The spectrophotometric analysis assessed the encapsulation efficiency at a wavelength of 224 nm, yielding an encapsulation efficiency of 86.01% (Fig. 1A). Figure 1B illustrates the drug release profile of Uro-B from GQD@MSN NPs, showing a drug loading capacity of 24%. Also, it presents a calibration curve for Uro-B (y = 0.3797x + 0.0807, R² = 0.9994), confirming the analytical method’s reliability. Figure 1B shows Uro-B’s in vitro cumulative release over 72 h, displaying a biphasic pattern: an initial burst of about 20% in the first 10 h, followed by a sustained release, achieving 49.5% cumulative release at 48 h and leveling off at about 54.3% after 72 h.

NP characterizations

DLS assay

DLS analysis revealed distinct characteristics for the two NP formulations. For GQD@MSN-Uro-B (Fig. 2A), the Z-average particle size was measured at 223.75 nm, with a polydispersity index (PDI) of 0.3820, indicating moderate uniformity. The mean intensity diameter was 289.52 nm, while the mean volume diameter was 334.91 nm, suggesting a range of particle sizes. The mean number diameter was recorded at 61.05 nm, and the zeta potential was found to be -22.59 ± 3.29 mV, reflecting good stability in suspension. The mean electrophoretic mobility was − 1.73 ± 0.25 mV.

In contrast, MSN-Uro-B @GQD (Fig. 2B) exhibited a larger Z-average particle size of 241.02 nm and a similar PDI of 0.3860. The mean intensity diameter was 312.90 nm, and the mean volume diameter was 367.67 nm, indicating a broader size distribution. The mean number diameter was slightly higher at 68.75 nm, with a zeta potential of -14.99 ± 5.31 mV, suggesting reduced stability compared to GQD@MSN-Uro-B. The mean electrophoretic mobility for this formulation was − 1.16 ± 0.41 mV. Both formulations possess suitable characteristics for potential biomedical applications; however, GQD@MSN-Uro-B shows stability and particle size advantages, which could improve its effectiveness in therapeutic settings.

FESEM and TEM

Figure 3 provides a detailed morphological characterization of the synthesized nanoparticles. The FESEM image of GQD@MSN-Uro-B NPs (Fig. 3A), revealing well-dispersed, spherical particles with uniform size distribution and smooth surfaces, indicates successful synthesis and coating of MSNs with GQDs and Uro-B loading. The FESEM image of MSN-Uro-B@GQD (Fig. 3B), where the NPs appear slightly less uniform and more sparsely distributed, possibly reflecting surface modification or loading sequence differences. Figure 3C presents the TEM image of GQD@MSN-Uro-B NPs, confirming their spherical morphology and nanoscale size (well below 100 nm), as well as the presence of a darker core-shell structure that suggests successful encapsulation of GQDs and Uro-B within the MSN matrix.

FTIR spectroscopy

As illustrated in Fig. 4, FTIR spectroscopy was employed to analyze the functional groups in various NP formulations. The spectra for GQD@MSN-Uro-B exhibited characteristic peaks at 3403.7 cm⁻¹ (O–H stretching), 2958.00 cm⁻¹ (C–H stretching), and 1641.82 cm⁻¹ (C=O stretching), among others, indicating the presence of hydroxyl, aliphatic, and carbonyl groups. The peaks at 1512.15 cm⁻¹ and 1388.88 cm⁻¹ suggest the presence of aromatic compounds and C–H bending vibrations, respectively (Fig. 2A). For MSN-Uro-B @GQD, the FTIR spectrum displayed a broader range of peaks, including significant absorptions at 3387.06 cm⁻¹ and 3301.61 cm⁻¹ (O-H stretching) and 2925.31 cm⁻¹ (C–H stretching). Notably, the peak at 1703.37 cm⁻¹ corresponds to C=O stretching. In comparison, additional peaks at 1625.84 cm⁻¹ and 1609.53 cm⁻¹ indicate the presence of aromatic structures and amide functionalities (Fig. 2B). This formulation showed a more complex spectrum, suggesting the successful incorporation of Uro-B into the MSN matrix. The MSN Blank sample revealed peaks at 3436.93 cm⁻¹ (O–H stretching) and 2941.66 cm⁻¹ (C–H stretching), with other notable peaks at 1635.66 cm⁻¹ and 1510.68 cm⁻¹, confirming the presence of surface silanol groups and residual organic moieties (Fig. 2C). The GQD spectrum demonstrated peaks at 3421.77 cm⁻¹ (O–H stretching), 2918.39 cm⁻¹ (C–H stretching), and 1693.22 cm⁻¹ (C=O stretching), indicative of the chemical structure of GQD with functional groups that may facilitate interactions with other components (Fig. 2D). In the GQD-NHS-EDC-MSN formulation, peaks at 3443.45 cm⁻¹ (O–H stretching) and 2949.83 cm⁻¹ (C–H stretching) were observed, along with a peak at 1632.65 cm⁻¹, suggesting successful conjugation of NHS and EDC groups to the MSN surface (Fig. 2E). The Uro-B spectrum displayed significant peaks at 3292.90 cm⁻¹ (O–H stretching), 3076.5 cm⁻¹ (aromatic C–H stretching), and 1701.04 cm⁻¹ (C=O stretching), confirming the presence of the drug’s characteristic functional groups (Fig. 2F). Finally, the MSN-Urolitin B formulation showed peaks at 3387.06 cm⁻¹ (O–H stretching), 3298.42 cm⁻¹ (N–H stretching), and 2929.40 cm⁻¹ (C–H stretching), indicating successful encapsulation of Urolitin B within the MSN structure (Fig. 2G). Overall, the FTIR analysis confirms the successful synthesis and functionalization of the NP formulations, highlighting the presence of key functional groups essential for their potential biomedical applications.

Cytotoxicity assay

The MTT assay results reveal significant differences in cytotoxic effects between the tested formulations on MCF7 breast cancer cells and normal HDF cells. GQD@MSN-Uro-B exhibited the most potent cytotoxicity against MCF7 cells (Fig. 5A), with an IC50 of 10 µg/mL, indicating high potency, while showing much lower toxicity toward HDF cells (IC50 = 231.2 µg/mL) (Fig. 5F), which demonstrates its selectivity for cancer cells. MSN-Uro-B@GQD also reduced MCF7 viability but with a higher IC50 of 83.19 µg/mL (Fig. 5B), indicating less potency than GQD@MSN-Uro-B. Urolithin B alone had an IC50 of 23.98 µg/mL for MCF7 cells (Fig. 5C) and over 500 µg/mL for HDF cells (Fig. 5G), suggesting that free Uro-B is less toxic to normal cells. The blank GQD@MSN formulation showed much less cytotoxicity toward both cell types, with an IC50 of 211.93 µg/mL for MCF7 cells (Fig. 5D) and greater than 500 µg/mL for HDF cells (Fig. 5H), confirming the safety of the carrier system. As a positive control, Tamoxifen demonstrated an IC50 of 15 µg/mL for MCF7 cells (Fig. 5E), which was less cytotoxic than GQD@MSN-Uro-B.

Annexin V-FITC/PI assay

The results illustrated in Fig. 6 reveal an apparent dose-dependent increase in apoptotic activity in MCF7 cancer cells treated with GQD@MSN-Uro-B NPs. Untreated control cells showed the lowest apoptosis rates, with 8.33% in early apoptosis and 3.78% in late apoptosis (Fig. 6A). Treatment with 8 µg/mL of the nanoparticles resulted in a 25.5% increase in early apoptosis and a 6.21% increase in late apoptosis (Fig. 6B). At a 10 µg/mL concentration, early apoptosis rose to 33.0%. In comparison, late apoptosis increased to 20.6% (Fig. 6C). Finally, at the highest concentration tested, 12 µg/mL, early apoptosis reached 37.4%. Late apoptosis surged to 39% (Fig. 6D). These findings suggest that GQD@MSN-Uro-B NPs are highly effective at inducing apoptosis in cancer cells concentration-dependent, indicating their potential as a targeted therapeutic for cancer treatment.

AO/PI staining

AO/PI staining of MCF7 cells treated with GQD@MSN- Uro-B NPs shows distinct fluorescence patterns based on dosage. The untreated control cells display uniform green fluorescence, indicating healthy cells (Fig. 7A). At 8 µg/mL, some cells reveal both green and orange/red fluorescence, indicating viable and early apoptotic cells (Fig. 7B). The 10 µg/mL concentration shows increased orange/red fluorescence, suggesting more late-apoptotic or necrotic cells (Fig. 7C). At 12 µg/mL, many cells exhibit orange/red fluorescence, indicating high levels of apoptosis and cell death (Fig. 7D).

Gene expression assay

Treatment of MCF7 cancer cells with GQD@MSN-Uro-B NPs resulted in a significant increase in the TNF biomarker, with expression rising from 0.92 ± 0.008 at 8 µg/mL to 1.13 ± 0.01 and 1.14 ± 0.012 at 10 and 12 µg/mL, respectively (Fig. 8A; P < 0.05). Additionally, the apoptosis-related biomarker caspase-9 (CAS-9) showed a dose-dependent increase, rising from 0.85 ± 0.008 at 8 µg/mL to 2.56 ± 0.01 and 3.3 ± 0.012 at 10 and 12 µg/mL (Fig. 8B; P < 0.001). Similarly, the cell cycle regulator p21 increased from 0.87 ± 0.008 at 8 µg/mL to 1.32 ± 0.012 at 12 µg/mL (Fig. 8C). These findings suggest that GQD@MSN-Uro-B NPs effectively modulate key biomarkers related to apoptosis and cell cycle regulation in MCF7 cells, indicating potential as an anti-cancer therapeutic.

Antioxidant capacity assay

The antioxidant capacity of GQD@MSN-Uro-B NPs was evaluated based on their ability to scavenge ABTS and DPPH free radicals (Fig. 9). The results indicated a significant concentration-dependent increase in scavenging activity for both assays (P < 0.001). Specifically, the ABTS assay demonstrated a 76% inhibition at a concentration of 2000 µg/mL, while the DPPH assay showed a 33.93% inhibition at the same concentration. These findings confirm that GQD@MSN-Uro-B nanoparticles exhibit notable antioxidant properties, particularly against ABTS radicals.

Targeted cancer therapies have significantly advanced over conventional treatments by concentrating on specific molecular targets that promote tumor growth²². Targeted therapies enhance treatment efficacy while reducing side effects by utilizing drug delivery systems (DDSs). Technologies such as nanoparticles, liposomes, and conjugated antibodies enable direct delivery to cancer cells, improving drug bioavailability and controlled release. This approach addresses drug resistance by engaging altered pathways in cancer cells and supports personalized medicine, allowing for tailored treatments and potential combinations for better outcomes. As research progresses, the integration of targeted therapies into clinical practice holds great promise for improving patient prognosis and quality of life²³.

The study presents compelling evidence regarding the anti-cancer potential of GQD@MSN- Uro-B nanoparticles, showcasing their effectiveness against the MCF7 cancer cell line through various assays. The unique combination of these two components in GQD@MSN-Uro-B NPs leverages their strengths while addressing limitations such as poor solubility and stability of free urolithins. DLS analysis indicates that GQD@MSN-Uro-B has superior stability and smaller particle size than MSN-Uro-B @GQD, which may enhance its therapeutic efficacy. FTIR spectroscopy confirmed the successful functionalization of the NPs. The cytotoxicity assays reveal a significant concentration-dependent effect, with GQD@MSN-Uro-B exhibiting a lower IC₅₀ (10 µg/mL) value against MCF7 cells while being less harmful to normal HDF cells, suggesting its selective targeting of cancer cells while sparing normal cells. It is necessary to explain that in the GQD@MSN-Uro-B formulation (Method 1), Uro-B is loaded onto pre-formed GQD-modified MSNs, allowing for better interaction with GQDs and the MSN matrix. This results in a more homogeneous distribution of Uro-B, facilitating efficient and sustained drug release. The unique properties of GQDs may enhance cellular uptake and increase cancer cell cytotoxicity through improved membrane interaction and increased ROS generation. In contrast, the MSN-Uro-B@GQD formulation (Method 2) loads Uro-B into MSNs before coating with GQDs, which can hinder immediate drug release and reduce direct interaction with cancer cells. The GQD coating may also slow drug release and limit ROS generation, decreasing Uro-B bioavailability at the target site. Consequently, GQD@MSN-Uro-B shows improved drug release, cellular uptake, and greater cytotoxicity against MCF-7 breast cancer cells compared to MSN-Uro-B@GQD, emphasizing the significance of nanoparticle design and drug loading sequence in optimizing therapeutic efficacy. In addition, the selectivity of GQD-modified MSNs for MCF-7 breast cancer cells, as opposed to non-cancerous cells, stems from several factors. GQDs’ small, lamellar structure allows them to enter cancer cell nuclei, disrupting DNA activity and inducing apoptosis selectively in cancer cells. Functionalizing GQDs with targeting ligands enhances their uptake by cancer cells that overexpress specific receptors. Additionally, cancer cells show higher metabolic activity and enhanced endocytosis, facilitating the internalization of GQD-modified MSNs. The mesoporous silica framework acts as a biocompatible carrier, promoting drug loading and controlled release, while GQDs generate ROS and target the nucleus. These combined features enable GQD@MSN nanocomposites to effectively kill MCF-7 cancer cells while minimizing toxicity to normal cells. Apoptosis assays further support these findings, demonstrating a marked increase in early and late apoptosis in treated cells. Additionally, gene expression analysis shows upregulation of critical biomarkers associated with apoptosis and cell cycle regulation, reinforcing the NPs’ role in inducing cancer cell death. We selected Caspase 9, p21, and TNF for gene expression analysis due to their roles as key markers in apoptosis and cell cycle regulation, which are essential for the anti-cancer effects of our nanoplatform. Caspase 9 is crucial in the intrinsic apoptotic pathway, indicating apoptosis in response to stress or chemotherapy. p21, a cyclin-dependent kinase inhibitor, is vital for G1/S checkpoint arrest, inhibiting cancer cell proliferation following DNA damage. TNF is a pro-inflammatory cytokine that triggers extrinsic apoptosis and influences the tumor microenvironment. Our findings are consistent with prior studies that have shown the ability of urolithins to modulate apoptosis-related genes, including BAX and BCL-2, thereby tipping the balance toward cell death in cancerous tissues²⁴. The antioxidant capacity of GQD@MSN-Uro-B is also noteworthy, as it effectively scavenges free radicals, adding another layer to its therapeutic profile. Oxidative stress is a well-established contributor to cancer progression, and the ability of these nanoparticles to scavenge free radicals adds a complementary mechanism to their anti-cancer activity. The results suggest that GQD@MSN-Uro-B NPs may create an antioxidant protection for healthy tissue, warranting further exploration in clinical settings. Overall, the use of GQDs to modify mesoporous silica nanoparticles is supported by their unique physicochemical properties, which greatly improve the delivery of Uro-B. GQDs demonstrate remarkable fluorescence, allowing for real-time tracking in biological systems, and possess high stability, ensuring prolonged circulation in the bloodstream. Additionally, their significant drug-loading capacity enables efficient encapsulation of Uro-B, thereby facilitating targeted therapy. Furthermore, the sequence in which GQDs are attached to MSNs is crucial in determining the surface chemistry and particle aggregation. For example, adding GQDs to MSNs may optimize surface interactions, enhance drug loading efficiency, and reduce aggregation. Conversely, attaching MSNs to GQDs could change the surface properties and potentially impact the overall effectiveness of the drug delivery system. This highlights the importance of GQDs in improving the therapeutic potential of Uro-B-loaded nanoparticles.

Previous studies have explored urolithins’ role in cancer therapy, particularly Uro-B²⁵. Urolithins are secondary metabolites formed from ellagic acid and ellagitannin, primarily found in foods such as pomegranates, berries, and nuts, through the action of gut microbiota²⁶. These compounds are categorized into three metabotypes based on the specific urolithins produced: metabotype A, which produces Uro-A; metabotype B, which produces Uro-A, IsoUro-A, and Uro-B; and metabotype 0, which makes none²⁷. Urolithins are noted for their enhanced bioavailability compared to ellagic acid and ellagitannin, achieving micromolar concentrations in plasma and various tissues, including the breast, colon, and prostate. Their anti-cancer effects are significant, as they regulate the expression of oncogenes, cell cycle mediators, tumor suppressors, and growth factor receptors, thereby contributing to their potential in cancer prevention and therapy^16,28,29. Additionally, urolithins exhibit anti-obesity, antimicrobial, and anti-inflammatory properties, highlighting their therapeutic relevance.

Urolithins, particularly Uro-B, have demonstrated significant anti-cancer effects in various cancer types, including prostate cancer. Research indicates that Uro-B induces apoptosis and inhibits proliferation in prostate cancer cell lines such as LNCaP and DU145. This dose-dependent effect leads to cell cycle arrest at the S and G2/M phases. Additionally, Uro-B treatment reduces prostate-specific antigen (PSA) and androgen receptor expression, inhibiting their interaction and transcriptional activity, which are crucial for tumor progression^30,31,32. Notably, Urolithin enhances the effects of bicalutamide in androgen-dependent cell lines, suggesting its potential to overcome resistance in castration-resistant prostate cancer (CRPC)^25,33. Overall, these findings position Uro-B as a promising candidate for prostate cancer treatment, warranting further clinical investigation.

In addition to its effects on prostate cancer, Urolithins exhibit significant anti-cancer properties in breast cancer, particularly in hormone-dependent cell lines. Both Uro-B and Uro-A show potent antiproliferative effects by inhibiting the proliferation of MCF-7aro cells, which overexpress aromatase³⁴. Their ability to inhibit aromatase positions them as potential therapeutic agents targeting estrogen synthesis, a critical factor for breast cancer growth. Furthermore, Uro-B demonstrates notable antiestrogenic effects, with lower IC₅₀ values in binding assays to estrogen receptors (ERα and ERβ) compared to many phytoestrogens, indicating its effectiveness in blocking estrogen signaling³⁵. In studies involving MDA-MB (estrogen-negative) and MCF-7 (estrogen-positive) cells, Uro-B exhibited decreasing antiproliferative potential, reinforcing its role in combating breast cancer growth. While phase II metabolites of Urolithins showed reduced activity, the aglycones retained significant effects, underscoring the importance of Uro-B in breast cancer prevention and treatment strategies³⁶.

Moreover, Urolithins show promising anti-cancer effects against endometrial cancer by inhibiting cell proliferation and potentially preventing metastasis³⁷. Uro-B effectively reduces the proliferation of endometrial cancer cells, similarly to Uro-A, through an ERα-dependent mechanism. At a concentration of 10 µM, Uro-B promotes cell cycle arrest at the G2/M phase by modulating key cell cycle regulators³⁷. Uro-A and Uro-B also influence gene expression related to cancer progression, acting as estrogen agonists. Notably, Uro-A decreases the activity and mRNA levels of Rac1 and PAK1, which are vital for cytoskeletal dynamics and cancer cell migration, indicating a preventive role against metastasis³⁸. These findings collectively highlight the therapeutic potential of Urolithins, particularly Uro-B, in endometrial cancer treatment.

Urolithins also exhibit significant anti-cancer effects against hepatocellular carcinoma (HCC) through various mechanisms. Uro-B effectively inhibits HCC cell proliferation, inducing cell cycle arrest at the G0/G1 phase in HepG2 cells and the S phase in Bel7402 cells³⁹. This antiproliferative effect is linked to the induction of apoptosis, evidenced by reduced Bcl-2 expression⁴⁰. In vivo studies demonstrate that Uro-B suppresses tumor growth in xenograft mouse models at 40 mg/kg. Additionally, Uro-B phosphorylates β-catenin, preventing its nuclear-to-cytoplasmic translocation and thus inactivating the Wnt/β-catenin signaling pathway, which is critical in cancer progression⁴⁰. These findings underscore Uro-B’s potential as a therapeutic agent for HCC.

The anti-cancer capabilities of Uro-B extend to colorectal cancer, where it induces apoptosis and cell cycle arrest⁴¹. Specifically, Uro-B (30 µg/mL) promotes apoptosis in HT-29 colon cancer cells by activating caspase 3 and upregulating p21, leading to G2/M phase arrest⁴². Furthermore, Uro-B inhibits mucin-type O-glycosylation in colon cancer cells, which correlates with decreased podoplanin (PDPN) expression and reduced cell migration and invasion⁴³. While Uro-B enhances the expression of detoxifying enzymes, it also induces CYP1B1, which may present undesirable effects in cancer therapy⁴⁴. These multifaceted mechanisms highlight Uro-B’s potential as a therapeutic agent in combating colorectal cancer.

Uro-B exhibits promising anti-cancer effects against bladder cancer, particularly in UMUC3 cell lines, characterized by cell cycle checkpoint abnormalities leading to genetic instability. Uro-B, Uro-A, and Uro-C reduce cell viability, demonstrating the most significant activity by inducing G2/M phase arrest⁴⁵. This arrest is attributed to Uro-A’s ability to inactivate the cyclin B1/cdc2 kinase complex, which is crucial for the G2/M transition⁴⁶. Furthermore, Uro-A has been shown to decrease the phosphorylation of p-Akt and ERK 1/2, indicating its potential to inhibit the PI3K/Akt and ERK pathways, vital for cancer cell proliferation and survival^47,48. These findings suggest that Uro-B could be a valuable therapeutic agent in managing bladder cancer. Furthermore, Uro-B shows promise as a potential treatment for osteosarcoma by exhibiting antimetastatic, antiproliferative, and apoptotic effects on the MG-63 OS cell line. The study revealed that Uro-B induces late apoptosis, necrosis, and G2/M cell cycle arrest while increasing the expression of the tumor suppressor gene p53 and the pro-apoptotic gene Bax. Additionally, Uro-B inhibits cell migration and metastatic behavior by downregulating MMP-2 and MMP-9 expression. These findings suggest that Uro-B may serve as a novel, nontoxic natural compound for osteosarcoma therapy, although further in vivo studies are needed²⁴. The molecular targets of Uro-B, such as BAX/BCL-2 and cell cycle regulators, play a critical role in mediating apoptosis and cell cycle arrest in cancer cells. The enhanced absorption and delivery efficiency of GQD@MSN-Uro-B NPs significantly amplifies the bioavailability of Uro-B, allowing for more effective modulation of these targets. By facilitating targeted delivery, GQD@MSN NPs ensure that higher concentrations of Uro-B reach the intended sites of action within the cells, thereby promoting the activation of pro-apoptotic pathways and inhibiting anti-apoptotic mechanisms. This innovative formulation not only highlights the therapeutic potential of Uro-B but also underscores the importance of nanoparticle carriers in enhancing the efficacy of cancer treatments.

This study presents several limitations that warrant consideration for future research. While the anti-cancer effects and antioxidant properties of GQD@MSN-Uro-B NPs were evaluated in vitro, further investigations are needed to assess their pharmacokinetics and biodistribution in vivo. Understanding how these nanoparticles behave in a biological system will provide crucial insights into their therapeutic efficacy and safety profiles. Additionally, conducting experiments in xenograft tumor models could offer a more comprehensive understanding of the potential clinical applications of GQD@MSN-Uro-B in breast cancer treatment. These studies would help elucidate the nanoparticles’ mechanisms of action, optimize dosing regimens, and evaluate their long-term safety and effectiveness, ultimately bridging the gap between laboratory findings and clinical practice.

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Authors and Affiliations

1. Department of Biology, Ne.C., Islamic Azad University, Neyshabur, Iran

   Bahareh Nezhadhossein, Akbar Safipour Afshar, Fatemeh Saeid Nematpour & Mahnaz Mohtashami

2. Department of Biology, Ma.C., Islamic Azad University, Mashhad, Iran

   Masoud Homayouni Tabrizi

Contributions

Bahareh Nejadhossein: Investigation, Methodology and Writing-Original draft. Akbar Safipour Afshar, Masoud Homayoni Tabrizi, Fatemeh Saeid Nematpour, Mahnaz Mohtashami1: Supervision, Data curation, Conceptualization Software, Validation, and Writing Reviewing.

Corresponding authors

Correspondence to Akbar Safipour Afshar or Masoud Homayouni Tabrizi.

Competing interests

The authors declare no competing interests.

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